1. Field Of The Invention
The present invention relates to the recombinant expression of analog subunits of cholera exotoxin, and to vaccines based on such analogs. More particularly, genetically engineered modifications of the exotoxin provide analogs of cholera toxin having the capability to elicit a protective response with reduced or essentially no catalytic activity which can contribute to the reactogenicity of cholera vaccines.
2. Description Of The Art
The term "cholera" refers to the disease caused by infection with the etiologic agent Vibrio cholerae, most commonly occurring in geographical areas where poor hygienic conditions prevail. Cholera remains a major cause of morbidity and mortality in many parts of the world(1,2). Experience has shown that contraction of the disease usually confers long-lasting protection against subsequent exposure to the etiologic agent(3). Consequently, considerable effort has been devoted to the development of a vaccine that would be similarly protective. A parenteral whole cell cholera vaccine has been produced, but some no longer regard it as useful, particularly for young children who are at greatest risk from the disease(1).
As for many other infectious diseases, a biological exotoxin (in this case, "cholera toxin" or "CTX") encoded by the genome of the infectious agent and secreted by it, contributes significantly to the ability of the microorganism to colonize the infected host(4). Moreover, exposure to the toxin causes severe diarrhea and vomiting which result in dehydration, a life-threatening condition of the disease(3,5). These experiences suggest that a vaccine which elicits an immunologic response (e.g., antibodies) sufficient to neutralize the toxin would thus significantly help to prevent or reduce bacterial colonization and attendant symptoms such as diarrhea and vomiting. Thus, substantial effort has been applied toward developing a vaccine containing a non-toxic analog of the toxin, i.e., a "toxoid" (1,3-13). It is known that cholera toxin is a multi-subunit macromolecule consisting of a subunit termed "A", containing a catalytic region called "A1" which ADP-ribosylates G-proteins in target cells, and a "B" oligomer which binds the holotoxin to the target cells(6). Non-toxic analogs of cholera toxin have been produced for purposes of vaccine development by various means. These methods include chemical treatment of the holotoxin or toxin subunits, deletion of the A subunit and use of the remaining B oligomer, and synthesis or isolation of peptide fragments of toxin subunits(1,3-13).
In recent years, efforts have turned toward the development of oral vaccines, with two approaches apparently having received the most attention. One of these approaches is based on the use of killed V. cholerae (i.e., chemically- or heat-inactivated), alone, or supplemented with the B oligomer of cholera toxin(1,11,12). This approach has been found to produce incomplete protection, particularly in young children(12). The other approach involves the use of living, but attenuated, strains of V. cholerae which fail to produce the A1 subunit of the toxin(13). Vaccines of this kind have provided greater levels of protection, but until recently have also been associated with unacceptable intestinal side-effects. A recently-developed vaccine based on V. cholerae strain CVD 103-HgR, in which the gene encoding the A subunit is omitted, appears to be better tolerated, at least in adults(13). However, to our knowledge, this vaccine has not been tested in children or in large-scale clinical trials.
Recent studies on the nature of cholera toxin have provided insights concerning its structure that may have application in vaccine development based on a recombinant approach. It is known that naturally-ocurring subunit A is synthesized in V. cholerae as a preprotein(14), which is subsequently cleaved to proteolytically remove a signal peptide sequence of approximately 2,160 kDa. Further post-translational processing yields an amino-terminal polypeptide of approximately 21,817 kDa (subunit A1) and a carboxyl-terminal polypeptide of approximately 5,398 kDa (subunit A2), which are linked by a disulfide bridge(6,15,16); reduction of the disulfide bond is believed necessary for catalysis of the ADP-ribosyltransferase reaction (6,15,16). Likewise, the B subunit is synthesized as a preprotein which is subsequently cleaved by protease to remove a signal peptide. The genes, or cistronic elements, for the A1, A2 and B subunits of cholera toxin have all been fully sequenced and described in the literature(16).